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SEMINAR ON :

There is no greater object of wonder, there is no greater inning of beauty than the ordered series and Linnaeus. :

There is no greater object of wonder, there is no greater inning of beauty than the ordered series and Linnaeus. organized complexities of life

Signaling is an essential part of communication :

Signaling is an essential part of communication Primary signaling
Secondary signaling Secondary signaling molecules
Cyclic nucleotides
Phospho-lipids
Ca2+
British biologist Sydney Ringer first demonstrated the biological significance of calcium. In 1883
Hundred year later it is Rasmussen elucidated the secondary signaling role of Ca2+

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UNIQUE CALCIUM:
It is believed that calcium stores in cells could have originated primarily for the purpose of defence against the ‘calcium holocaust’ (Campbell 1988).
Since the intracellular level of Mg2+ ion is already high, a rapid and large (>10-fold) increase is not possible for this ion
Ca2+ ion has the maximum affinity for carboxylate oxygen, 0.82% of (Glu + Asp) over(Asn + His + Gln) amino acids. The important thing is that this translates into an excess of 77860 (Glu + Asp)
Ca2+ binds to and dissociates from a protein 100-fold or so faster than Mg2+ (Ochiai 1991)

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MODEL FOR Ca2+ REGULATION

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Stimuli
Signal
Signal coding
Signal decoding
Gene expression
Response

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An Overview of Calcium Regulation in Cells. Cytosolic calcium concentration is lowered by the actions of the ER calcium pump, the plasma membrane calcium pump, sodium-calcium exchangers, and the mitochondria. Calcium concentration increases in the cytosol because of the opening of calcium channels in the plasma membrane and the release of calcium through the lnsP3 or ryanodine receptor channels in the ER membrane.

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Hyperosmotic Stress Elicits a Transient Ca2+cyt
In hyperosmotic stress nonselective cation channel in cell membrane opens
Increase in cytoplasmic calcium potentiates the ROS production in Mitochondria
In animals this process is associated with respiratiory disorders
In plant yet to be characterized (Susana et al., 2002)

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After 10 sec the calcium wave was observed in rhizoids of fucus
The speed of calcium wave as estimated as 15 µm/s
(Goddard et al., 2000) Calcium wave initiates in hyperosmotic condition

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Mitochondria contributes to intracellular sink for calcium
Increase in calcium was observed by mitochondrial calcium tracker X-rhod-EF Reactive oxygen species (ROS) production was monitored as oxidation
of 5- (and 6-) chloromethyl-2,7-dichlorodihydrofluorescein diacetate
(CM-DCFH2-DA)

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Proposed Signaling Pathway during Hyperosmotic Stress in Fucus.
Osmotic change is sensed by an unidentified osmosensor (1), which induces DPI-sensitive ROS production within 10 s (2). H2O2 is produced at the exterior side of the plasma membrane and may be involved in cell wall strengthening. H2O2 also diffuses into the cell, leading to a localized peripheral intracellular increase. Localized external H2O2 production also increases Ca2 channel activity (3). Downstream events include Ca2 wave propagation during the subsequent 10 to 60 sec (4), followed by Ca2 m increase (5) and mitochondrial ROS production (6). FCCP, carbonylcyanide p-trifluoromethoxyphenyl hydrazone.

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Calmodulin is a primary Ca2+ binding protein in cells
Involved in the signal relay mechanism of Ca2+ signaling
Have four Ca2+ binding sites separated by a 2 nm long helix
After ca2+ binding, it under go conformational change
And wraps around the protein to which it is destined to activate

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Structure of Ca2+binding protein Calmodulin (left), sites of Ca2+ binding on four loops of the protein (middle), details of single loop harboring calcium ion (right).

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Calmodulin binding proteins in plants are
Myosin V, Kinesin, NAD+ kinase, Glutamate decarboxylase Protein kinases, and Ca2+ ATPases, many more are still unidentified.

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Nitric oxide production is responsible for success of HR (Zemojtel et al., 2006)
In plant Nitrate Reductase is the potential nitric oxide producer but is it the responsible candidate for HR Nitric oxide production? A, NO generation in the absence (white bar) and presence (gray bar) of the NR inhibitor (1 mM) NaN3. No, then what?

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This says may be NOS or similar enzyme in plant have role in HR
NOS like activity proved in infected plant extracts B, NO generation in the absence (white bar) and presence (gray bar) of the NOS inhibitor (20 mM) diphenyleneiodonium chloride(DPI).

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This NOS activity is dependent on calcium concentration There is a link between the calcium, Calmodulin and NOS in the plant pathogen response signaling cascade (Ali et al., 2007) C, Effect of the Ca2+ chelator EGTA on NO generation.

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(Wei Ma et al., 2008) D, All assays included 1 mM NaN3. In the presence of this NR inhibitor, NO generation was monitored in the presence and absence of 1 mM Ca2+ and 1 mM CaM. E, In the presence (left) or absence (right) of 1 mM CaM, NO generation was monitored in the absence (white bars) or presence (gray bars) of 200 mM W7. All assays in A to C included 1 mM Ca2+ and 1 mM CaM.

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Co- infiltration of Calmodulin antagonist with pathogen impairs the hyper sensitive reaction(Di fluoperazine dihydrochloride-w7)
But Calmodulin antagonist have no effect on the sustained calcium rise
The calmodulin antagonist blocks the Nitric Oxide generation NO binding fluorophore of DAF2DA is generated only within cells upon cleavage of the ester linkage; hence, the dye responds only to intracellular NO. NO-specific fluorescent dye 4,5-diaminofluorescein diacetate

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The CaM antagonist W7 prevents HR in wild-type plants. A, Inoculated regions of the leaves are highlighted by boxes. As shown in the top three panels, HR development becomes visible at 13 h after inoculation

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Loss-of-function cml24-4 plants show an impaired HR phenotype. A, Inoculated areas of the leaves are highlighted by boxes. In the top two panels, HR development B, Measurement of ion leakage in response to inoculation Hypersensitive reaction was evident in wild type

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FUTURE PERSPECTIVES
As Ca2+ signaling is essential in pollen tube growth, it must have role in self incompatibility and male sterility.
Thorough understanding of Ca2+ signaling have a good potential to tackle the problems of biotic and abiotic stresses
A new field called kinomics is on its way to decode the role of different kinases in Ca2+ signaling
Can't we think of deriving sustainable solution for CO2 dependent global worming by unraveling Ca2+ signaling in CO2 sinking marine flora?